Interleukin-2 (IL-2) is a T-cell derived factor that amplifies the response of T cells to any antigen by stimulating the growth of the T cells. Thus, IL-2 is a critical T-cell growth factor which plays a major role in the proliferation of T cells that occurs subsequent to antigen activation, this proliferation resulting in the amplification of the number of T cells responsive to any particular antigen. IL-15 can generally substitute for IL-2 to exert most, if not all, of these activities (Bamford et al., 1994).
The high affinity (Kd:10.sup.-11 M) IL-2 receptor (IL-2R) is composed of at least three non-covalently associated IL-2 binding proteins: the low affinity (Kd:10.sup.-8 M) p55 (.alpha. chain) and the intermediate affinity subunits (Kd:10.sup.-9 M) p75 (.beta. chain) and p64 (.gamma. chain) (Smith, K. A., 1988; Waldmann, T. A., 1993). Proliferative signals for the T cells are delivered through high affinity IL-2 receptors consisting of all three subunits, but not via the low affinity site (Robb, R. J. et al., 1984; Siegal, J. P. et al., 1987; Hatakeyama, M. et al., 1989). IL-2R.alpha., IL-2R.beta., and IL-2R.gamma. chains have 13, 286 and 86 amino acid intracytoplasmic domains, respectively.
IL-15, a cytokine with many IL-2-like activities, also utilizes the IL-2R.beta. as a part of its receptor complex (Giri et al., 1994). This IL-2R.beta. dependent signaling process is fundamental to the cellular effects induced by the binding of IL-2 to its receptor (IL-2R) as well as the effects induced by the binding of IL-15 to its receptor. The IL-2R.beta. and .gamma. chains, but not the .alpha. chain, are essential for IL-2- as well as IL-15-mediated signal transduction (Nakamura, Y. et al., 1994). The 64 kDa IL-2R.gamma. chain protein is rapidly phosphorylated on tyrosine residues after stimulation with IL-2. The .gamma. chain has also been shown to be a part of other receptor complexes such as the receptor for IL-4 and IL-7 (Noguchi, M. et al., 1993; Russell, S. M. et al., 1993). Absence of the .gamma. chain leads to a severe combined immunodeficiency disease in humans (Noguchi, M. et al., 1993). IL-2R.gamma. contains sequences from positions 288 to 321 homologous to the Src homology region 2 (SH2) that can bind to phosphotyrosine residues of some phosphoproteins. Another molecule, designated pp97, has been suggested to be the tyrosine kinase physically associated with the IL-2R.gamma. chain (Michiel, D. F. et al., 1991).
An analysis of cells transformed with a series of IL-2R.beta. chain deletion mutants identified a 46 amino acid serine and proline rich intracytoplasmic region of the IL-2R.beta. chain (a.a. 267-312), which is crucial for growth promoting signal transduction (Hatakeyama, M. et al., 1989). This same region is crucial for promoting IL-15 mediated effects. Upon stimulation with IL-2, enzymatically active protein tyrosine kinases and, as the laboratory of the present inventors has previously shown (Remillard, B. et al., 1991), the novel lipid kinase, phosphatidyinositol-3-kinase activity blocks proliferation. Cells that express wild-type IL-2R.alpha. and .gamma. chains and mutant IL-2R.beta. chains lacking this 46 a.a. region bind and internalize IL-2, but fail to proliferate in response to IL-2 (Hatakeyama, M. et al., 1989). An identical set of circumstances pertains to IL-15 responses. Although the intracytoplasmic domain of the IL-2R.beta. and .gamma. chains lacks a protein tyrosine kinase consensus sequence, several cellular proteins are phosphorylated upon tyrosine residues following IL-2 stimulation (Benedict, S. H. et al., 1987; Ferris, D. K. et al., 1989; Saltzmann, E. M. et al., 1988; Asao, H. et al., 1990; Mills, G. B. et al., 1990; Merida, I. and Gaulton, G. N., 1990). IL-2 induced protein tyrosine kinase activity is due, at least in part, to activation of the p56.sup.lck (lck), a src-family protein tyrosine kinase. Controversy exists as to whether the serine/proline rich (Fung, M. R. et al., 1991) or an adjacent tyrosine rich "acidic" region (Hatakeyama, M. et al., 1991) of the IL-2R.beta. chain is the lck binding site.
IL-2 also stimulates phosphorylation on serine residues of several proteins (Turner, B. et al., 1991; Valentine, M. V. et al., 1991). Raf-1, a serine/threonine kinase, has been identified as a likely signal transducing element for several growth factor receptors (Carroll, M. P. et al., 1990; Morrison, D. K. et al., 1988; Baccarini, M. et al., 1991; Kovacina, K. S. et al., 1990; Blackshear, P. J. et al., 1990; App, H. et al., 1991). The Raf-1 molecule has a molecular weight of 74 kD and can be divided into 2 functional domains, the amino-terminal regulatory half and the carboxy-terminal kinase domains (for review see Heidecker, G. et al., 1991). Raf-1 has been identified as a crucial signal transducing element for ligand activated EPO receptors (Carroll, M. P. et al., 1991). The IL-2R.beta. chain and EPO receptors belong to the same family of receptors and share homologies within their cytoplasmic domains (D'Andrea, A. D. et al., 1989). Stimulation of the IL-2R results in the phosphorylation and activation of cytosolic Raf-1 serine/threonine kinase. IL-2R stimulation leads to a 5 to 10 fold immediate/early induction of the c-raf-1 mRNA expression on freshly isolated, resting T cells (Zmuidzinas, A. et al., 1991) and results in up to a 12-fold increase in Raf-1 protein expression. In addition, a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increases through G1.
Enzymatically active Raf-1 appears in the cytosol of IL-2 stimulated CTLL-2 cells (Hatakeyama, M. et al., 1991) and human T blasts (Zmuidzinas, A. et al., 1991). Following IL-2 stimulation, cytosolic Raf-1 molecules are phosphorylated on tyrosine and serine residues (Turner, B. et al., 1991). The laboratory of the present inventors have studied the signaling pathway by which IL-2 signals T cells to begin dividing. In these studies Raf-1 was identified in immunoprecipitates of the IL-2R.beta. chain, suggesting that Raf-1 may be involved as an important element in IL-2 signaling. Further, it was determined that prior to IL-2 stimulation, enzymatically active Raf-1 molecules are physically associated with the IL-2R.beta. chain and that following stimulation with IL-2, a protein tyrosine kinase phosphorylates Raf-1 thereby leading to translocation of Raf-1 from the IL-2 receptor into the cytosol (Maslinski, W. et al., 1992). Moreover, dissociation of enzymatically active Raf-1 from the IL-2R.beta. chain, but not maintenance of IL-2R associated kinase activity, is completely abolished by genistein, a potent tyrosine kinase inhibitor (Maslinski, W. et al., 1992). The above-noted suggested requirement of Raf-1 for IL-2 signaling has been supported by evidence showing that by blocking Raf-1 expression, IL-2 could not induce T cell proliferation in the absence of Raf-1. Thus, from the afore-mentioned, it is widely accepted that activation of the Raf-1 serine/theonine kinase is critical for IL-2-mediated T-cell proliferation (see also Riedel et al., 1993).
Prior to IL-2 stimulation, several serine, but not tyrosine nor threonine, residues of the IL-2R.beta. chain are phosphorylated (Asao, H. et al., 1990). IL-2 induces rapid (i.e., within 10-30 min) phosphorylation of additional serines, tyrosines and threonines (Asao, H. et al., 1990; Hatakeyama, M. et al., 1991). Tyr 355 and Tyr 358 are major, but not exclusive, tyrosine phosphorylation sites of IL-2R (catalyzed by p561.sup.lck in Vitro (Hatakeyama, M. et al., 1991)). The phosphorylation sites of the IL-2R.beta. chain may play an important role in IL-2R.beta. chain signal transduction and interactions with accessory molecules (like p561.sup.lck and Raf-1).
Phosphorylation of Raf-1 has also been demonstrated in a human T cell line following CD4 cross-linking. Activation of Raf-1 has also been observed following TCR/CD3 complex stimulation by CD3 or Thy 1 cross-linking as well as an approximately four fold increase in c-raf-1 mRNA. In this case, Raf-1 phosphorylation occurs only on serines and is not observed if PKC had been down regulated. It is interesting to note in this context that GTPase-activating protein (GAP) activation and, consequently, Ras induction following TCR stimulation is also PKC mediated (Downward, J. et al., 1990).
However, the precise residues that form the contact points of p56.sup.lck tyrosine kinase, and PI-3-kinase to the IL-2R.beta. chain have not been established. Indeed, two groups (Greene and Taniguchi) have utilized grossly truncated IL-2R.beta. cDNA transfectants to analyze the binding sites of the IL-2R to lck (Hatakeyama, M. et al., 1991; the Greene group; Turner, B. et al., 1991; the Taniguchi group). Although they used essentially the same techniques and reagents, the conclusions of these studies are conflicting. It is possible that the use of drastically truncated mutants may result in conformational changes in the expressed protein that confound attempts to precisely map the residue to residue contact points required for ligand to ligand interaction. Moreover, recent data from Greene's group is more in line with Tanaguchi's data (Williamson, P. et al., 1994). However, the model cell line used by both laboratories (Baf/3) has been shown to signal differently than a T cell line CTLL2 (Nelson, B. H. et al., 1994). Thus, it is not completely clear which portions of the IL-2R.beta. chain are of most importance to normal T cells.
The recent characterization of so-called "knockout" mice for IL-2 (i.e., mice which lack IL2) has shown that about 50% die by nine weeks of age (Schorle, H. et al., 1991). Although these mice appear to be phenotypically normal and can mount some cell-mediated responses (Kundig, T. M. et al., 1993), they ultimately develop inflammatory disease. Recently, it has been suggested that the reason the mice are still relatively normal is that there is an additional cytokine (IL-15) that signals through the IL-2 receptor .beta. and .gamma. chains. Thus, there may be some compensation by IL-15 in these mice for the lack of the IL-2 molecules. On the other hand, deficiency of the IL-2R.gamma. chain in humans leads to a severe combined immunodeficiency, characterized by the near absence of both mature and immature T cells (Noguchi, M. et al., 1993). Further support for the importance of IL-2 in vivo comes from studies utilizing anti-IL-2 antibodies. Marked immunosuppressive effects in both transplantation and autoimmune models have been obtained by using anti-IL-2R.alpha. monoclonal antibodies (Strom, T. B. et al., 1993). Clinical efforts with similar anti-human IL-2R.alpha. antibodies (produced in mice as monoclonal antibodies) showed some efficacy but this was limited by a rapid immune response in the human patients to the murine monoclonal antibody, i.e., human-anti-mouse antibodies (HAMA) were produced in the patients a short time after treatment with the mouse-anti-human IL-2R.alpha. monoclonal antibodies.
Members of the highly conserved 14-3-3 protein family, first identified as abundant 27-30 kD acidic proteins in brain tissue (Moore et al., 1967) and later found in a broad range of tissues and organisms (Aitken et al., 1992), were recently found to be associated with the products of proto-oncogenes and oncogenes, such as Raf-1, Bcr-Ab1, and the polyomavirus middle tumor antigen MT (Fu et al., 1994; Reuther et al., 1994; Pallas et al., 1994; Irie et al., 1994; Freed et al., 1994). 14-3-3 appears to associate and interact with Raf-1 at multiple sites, i.e., amino terminal regulatory regions of Raf-1, kinase domain of Raf-1, zinc finger-like region of Raf-1, etc., with primary sites of interaction located in the amino-terminal regulatory domain (Fu et al., 1994; Freed et al., 1994). In comparing sequences of Bcr, Bcr-Ab1 and MT at sites of interaction with 14-3-3, cysteine- and serine-rich regions were found to be common elements and may be some of the determinants responsible for 14-3-3 binding (Morrison, 1994).
The results reported by Freed et al. (1994) and Irie et al. (1994) suggest that 14-3-3 modulates Raf-1 activity in yeast. For instance, Freed et al. (1994) found that over-expression of mammalian 14-3-3 proteins in yeast stimulated the biological activity of mammalian Raf-1, and observed that mammalian Raf-1 immunoprecipitated from yeast strains overexpressing 14-3-3 had three- to four-fold more enzymatic activity than Raf-1 from yeast strains lacking 14-3-3 expression. However, 14-3-3 proteins alone are not sufficient to activate the kinase activity of Raf-1, suggesting that 14-3-3 may be a cofactor involved in Raf-1 activation (Morrison, 1994; Freed et al., 1994). Because 14-3-3 constitutively associates with Raf-1 in vivo regardless of subcellular location or Raf-1 activation state or whether Raf-1 is bound to Ras (Fu et al., 1994; Freed et al., 1994), it is suggested that an alternate function of 14-3-3 may be a structural role in stabilizing the activity or conformation of signaling proteins (Morrison, 1994).
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